Introduction

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for certain malignant and non-malignant diseases. Unfortunately, in the Czech Republic, only approximately 15% of the children needing HSCT have a human leukocyte antigen (HLA) matched sibling donor (MSD) available. For the remaining 85%, unrelated donors (UDs) are searched for. For patients with rare HLA haplotypes, it is difficult to identify fully matched UD. Mismatched UD transplantation is associated with a high risk of graft-versus-host disease (GVHD) and transplant-related mortality. Cord blood or a haploidentical related donor graft can also be used, but this is associated with an increased risk of post transplant morbidity/mortality due to increased risk of GVHD, graft failure or infections.^{1, 2} Here we report our experience with GVHD prophylaxis using pre-transplant rabbit antithymocyte globulin (rATG) added to standard post transplant cyclosporin A (CsA) and methotrexate (MTX) in children.^{3, 4}

Methods

Patients and donors

Between January 2001 and December 2005, 88 children underwent allogeneic HSCT at our center using an UD. Rabbit ATG (Fresenius, dose and schedule below) for GVHD prophylaxis was used in 75 children (48 boys and 27 girls). Thymoglobuline once and Campath twice were used in three other patients, T-cell depletion in two including one with rATG and no serotherapy in four patients with advanced leukemia. Furthermore, patients receiving cord blood units were excluded from this cohort even though same rATG was used in them. These 75 patients (median age at transplant 12.8 years, range 0.3–20.5 years) received HSCT for malignant (n=54) or non-malignant disease (n=21) (Table 1). The grafts were obtained from HLA allele matched or partially mismatched UDs (age 21–57 years, median 32 years). HLA typing was performed at the high-resolution level (four digits) in A, B, Cw, DRB1 and DQB1 alleles. Most (72%) pairs were matched in 9 or 10/10, 17% in 8/10 and 11% in 7/10 alleles. Only one allele mismatch was accepted in A, B or DRB1 loci. Donors and patients were sex mismatched in 42 cases, with a female donor for a male recipient in 20 cases. Cytomegalovirus (CMV) immunoglobulin (Ig) G was detected pre-transplant in 28 (37%) donors and 44 (59%) recipients. The interval between the start of the UD search and the day of HSCT ranged from 49 to 944 days, median 132 days.

Table 1 - Primary diagnosis (n=75 patients).

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The study was approved by the local Ethics Committee and all parents signed informed consents.

Grafts

Filgrastim-mobilized peripheral blood stem cells (PBSCs) were used for 35 patients and bone marrow (BM) for 40 patients. The decision about the type of graft was made by the donor and/or local harvest center, and was based in part on transplant center preference. Characteristics of grafts are shown in Table 2. No graft manipulation other than plasma or erythrocyte depletion was carried out.

Table 2 - Characteristics of primary grafts and engraftment.

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Conditioning regimens, GVHD prophylaxis

Primary conditioning regimens varied according to primary disease (see Table 3). Rabbit ATG (Fresenius) was infused to all 75 patients over 1 h⁵ on days -4 to -1. The daily dose was 10 or 15 mg/kg (total 40 mg/kg in 70 patients; range 30–60 mg/kg). Combination of methylprednisolone (1–3 mg/kg), antihistamines and antipyretics was used as pre-medication before each infusion. CsA (used in 74/75 patients) was given two to three times daily in a 2-h infusion, initially 1.5 mg/kg/dose in malignant and 2.5 mg/kg/dose in non-malignant disease. The prophylactic dose of CsA was adjusted to maintain targeted blood levels, lower (80–150 ng/ml) for malignant and higher (200–250 ng/ml) for non-malignant diseases. MTX was given to 68/74 evaluable patients on days +1, +3 and +6. The first dose was 10 or 15 mg/m², and other doses were 10 mg/m². Leucovorine (15 mg/m²) was given as a single dose 24 h after each MTX dose. In one patient, CsA was substituted by mycophenolate mofetil (MMF) for elevated creatinine during conditioning regimen, and in six patients MTX was not used; it was substituted in four patients with steroids, in one with MMF, and no substitution in one patient. Acute and chronic GVHD were primarily treated with prednisone, CsA, tacrolimus, sirolimus or MMF.^{6, 7, 8} Acute and chronic GVHD were diagnosed and graded using established criteria.^{9, 10}

Table 3 - Characteristics of conditioning regimens used before first HSCT (n=75 patients).

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Chimerism analyses and minimal residual disease monitoring

Chimerism was assessed by using polymerase chain reaction (PCR)-based analyses of polymorphic variable number tandem repeats using peripheral blood leukocytes starting on day +14 and then once a week till day +100, with decreased frequency thereafter, especially in patients with stable full donor chimerism.¹¹ PCR assay of specific fusion genes and IgH/T-cell receptor (TCR) receptors according to the type of leukemia was used for minimal residual disease (MRD) monitoring pre- and post-HSCT. This was performed according to the criteria of the European study group on detection of MRD in acute lymphoblastic leukemia (ESG-MRD-ALL).^{12, 13} A marrow sample was obtained routinely from patients with hematological malignancy before HSCT, and at 28, 60 and 100 days and 6 and 12 months after transplantation. A complete hematological remission was defined as less than 5% blasts in the marrow aspirate.

Monitoring of viral infections

Epstein–Barr virus (EBV) and CMV reactivation was monitored weekly by quantitative PCR using whole blood. Results were normalized to 100 000 human genomic equivalents (g.e.) assessed by quantification of albumin gene. When samples obtained less than 1000 g.e., frequent monitoring was indicated, but pre-emptive therapy was not started.

Statistical methods

The two-sample Welch t-test for unequal variances was used to compare the means of continuous variables for two groups. The Fisher's exact test was used to examine the statistical significance of a relationship within a 2-by-2 table of categorized factors. The log-rank test was used to compare distributions of censored failure times between groups. P-values less than 0.05 were considered to indicate significance.

Results

Hematopoietic engraftment and chimerism

The median day of neutrophil engraftment (0.5 10⁹/l for 3 consecutive days) was 16 for PBSC and 20 for BM recipients (P<0.0001). The median day of platelet engraftment (20 10⁹/l for 7 consecutive days without platelet transfusion) was 23 for PBSC and 27 for BM recipients (P=0.01) as shown in Table 2. Complete donor chimerism was observed in 67 (92%) out of 73 evaluable patients, and was documented after a median of 21 days (range 14–180 days) with no difference between PBSC and BM recipients. There was no difference in the day of neutrophil or platelet engraftment between patient undergoing grafting from 7 to 8/10 and 9 to 10/10 allele-matched donors (median neutrophil engraftment 18 days in both groups, median platelet engraftment 24 and 23 days, respectively). Three patients developed graft failure; their donors were 8/10 (B, Cw), 8/10 (B, Cw) and 9/10 (A) allele-matched. Detailed characteristics of grafts and engraftment are shown in Table 2.

GVHD incidence and severity

Grade II–IV acute GVHD developed in 48 (65%) of 74 evaluable patients. The median day of onset was day 30 (range, 8–85). As shown in Table 4, grade III–IV acute GVHD was observed in two (3%) patients only. Overall incidence of chronic GVHD in 69 evaluable patients was 32%, 7% experienced limited and 25% extensive disease. There was no significant difference in the incidence or severity of acute GVHD (grades II–IV) or extensive chronic GVHD between patients receiving PBSC compared to BM recipients (Table 2). Even though there was no difference in the incidence of grade III–IV acute GVHD and limited chronic GVHD, both acute GVHD of grades II–IV and extensive chronic GVHD were higher (76 vs 59% for acute, 29 vs 20% for chronic) in patients undergoing grafting from 7 to 8/10 compared to those grafting from 9 to 10/10 allele-matched donors. However, this did not adversely affect the overall outcome.

Table 4 - Incidence and severity of acute and chronic GVHD.

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Viral complications

EBV reactivation (DNAemia above 1000 copies in 100 000 g.e.) was detected in 26 (29.4%) patients, and EBV lymphoproliferation/lymphoma developed in four (5%) patients. CMV reactivation (DNAemia above 1000 copies in 100 000 g.e.) was detected in 25 (28.4%) patients. CMV disease developed in six (7%) patients. Other viral diseases included BK virus (BKV) cystitis in eight patients (11%) and adenovirus pneumonitis in one patient (1%). The viral diseases were not fatal, except for one case of CMV pneumonia.

Relapse

Hematological relapse occurred in 12 of 54 (22%) patients following HSCT for malignant disease (see Table 5) at 153–842 days after transplant (median 314 days). Six patients subsequently died, retransplantation was performed in three patients (all surviving without leukemia), two patients are currently scheduled for retransplantation and one at the time of analysis lives in untreated extramedullary relapse. There is clear trend that even in our group of patients with ALL, the incidence of relapse was dependent on the level of pre-transplant MRD (see Table 5).^{12, 14} Donor lymphocyte infusion (DLI) directed according to the level of post transplant MRD (IgH/TCR >1 10^-4) or increasing mixed chimerism was given to 10 patients with malignancy. Initial dose of CD3+ cells was 1 10⁶/kg. In nine of them, there was no or only transient response. In none of those patients, DLI was used at the time of frank leukemia relapse.

Table 5 - Outcome of patients with ALL according to leukemia status before HSCT (22 patients, 23 HSCT).

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Survival

Overall survival was not statistically different (P=0.49) between patients transplanted using PBSC (82.9%) and BM (75.0%) (Figure 1). Overall survival was also not statistically different (P=0.76) between patients receiving grafts from donors HLA matched in 9–10/10 alleles (77.8%) and 7–8/10 alleles (80.9%) as shown in Figure 2. There was no significant difference (P=0.66) in overall survival and event-free survival (P=0.65) between patients undergoing HSCT for malignant and non-malignant disease (Figures 3 and 4).

Figure 1.

Overall survival (OS) in patients following HSCT using PBSC (n=40) or BM (n=35).

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Figure 2.

Overall survival (OS) in patients following HSCT from HLA-matched (9–10/10; n=54) or -mismatched UD (7–8/10; n=21).

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Figure 3.

Overall survival (OS) in patients with malignant disease (n=54) and non-malignant disease (n=21).

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Figure 4.

EFS in patients with malignant disease (n=54) and non-malignant disease (n=21).

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Sixteen patients died, 6/54 (11%) due to relapse and 10/75 (13%) due to transplant-related complications (7% before day 100). Six patients received a second graft from the same donor (median 155 days post transplant, range 22–502 days), three for graft failure/rejection and three for relapse. Four of the six patients are alive and well with median follow-up of 26 months (range, 16–49 months) following the second HSCT and two died with GVHD.

Discussion

The first important finding of this study is the similar outcome of transplantation using 7–8/10 and 9–10/10 allele-matched donors. This is contrary to previous studies in which HLA typing was not performed at the high-resolution level (four digits) in all typed loci. Serologically undisclosed HLA disparities account for the increased rate of post transplant complications. With more than 1300 alleles (A, B, Cw, DRB1) currently identified, high-resolution molecular typing techniques have to be applied to distinguish the extensive degree of allelic polymorphism of the HLA system. Whereas an HLA-ABDR serologically identical donor can be identified in the International Registry for >90% of the patients, only up to half of them can have a highly compatible donor if donor selection is based on allele level matching for HLA-A/B/Cw/DRB1/B3/B5/DQB1 loci among the Caucasian population. Our results suggest that a higher percent of pediatric patients could have an UD available, as even a 7/10 allele-matched donor (with limitation of maximum one allele mismatch in A, B or DRB1 loci) appears acceptable when ATG is used.

The second important finding of this study is the similar outcome of PBSC vs BM transplantation. After years of using PBSC for allogeneic HSCT, there is still ongoing discussion on whether PBSC or BM is the superior graft source. Perhaps, the reason for ongoing discussion is that studies comparing PBSC vs BM enrolled various patient populations in terms of primary disease, GVHD prophylaxis, donor type and level of HLA matching, etc.^{15, 16} These studies mostly confirm faster engraftment and higher incidence of chronic GVHD in recipients of PBSC from MSD. There are no published randomized studies comparing PBSC and BM in the UD settings. Two retrospective studies found significantly higher risk of chronic GVHD after PBSC transplantation and no survival advantage of PBSC transplantation over BM transplantation.^{17, 18} Compared to our cohort, only one-third of patients in the published retrospective studies received ATG or OKT3 pre-transplant. In our cohort (uniformly treated with ATG), we have observed similar incidence of extensive chronic GVHD and similar survival in PBSC and BM recipients. Thus, the use of ATG appears to eliminate the difference in the incidence of chronic GVHD. This may be important, as chronic GVHD is the major determinant of the quality of life of transplant recipients.¹⁷

Viral diseases remain a significant problem after unrelated HSCT.¹⁸ Theoretically the use of ATG might be associated with an increased incidence of viral disease, as ATG (persisting beyond day 0) could kill anti-viral T cells contained in the graft. However, the incidence of viral complications in our cohort was not unusually high. Incidence of CMV disease or EBV-LPD in our cohort was not higher compared to published data.^{19, 20, 21, 22}

Relapse is another significant problem of transplant recipients, which could be theoretically worsened by the use of ATG (through the killing of T cells mediating graft-versus-leukemia effect). However, the incidence of relapse in our cohort was not high. Moreover, the level of MRD before HSCT has been shown to be crucial for outcome. This fully correlates with published data where level of MRD proved to be the only significant risk factor in multivariate analysis and independent on the use of ATG.^{12, 13}

If our results are confirmed in prospective randomized studies, the use of ATG will possibly widen the choice of donors and grafts for children needing HSCT.

References

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Acknowledgements

We thank our collaborators from CPH (Czech Pediatric Hematology Working Group) for referring patients, national registries of donors in Pilsen and Prague, HLA laboratories namely in Institute of Hematology and Blood Transfusion and CLIP (Childhood Leukemia Investigation Prague) in Prague. We thank Jan Storek for editorial help. This work was partly supported by Grant IGA CR NR 8223/3.